Project Summary Cardiolipin (CL), the signature lipid of the mitochondrial membrane, is crucial for optimal mitochondrial function. The importance of CL is underscored by the fact that perturbation of CL metabolism due to mutation of the CL remodeling enzyme tafazzin (Taz) leads to the life- threatening genetic disorder, Barth syndrome (BTHS). While the clinical phenotypes of dilated cardiomyopathy and skeletal myopathy point to mitochondrial bioenergetic defects, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of amino acids and TCA cycle-associated metabolites. These studies suggest that CL plays an important role not only in oxidative phosphorylation but also in intermediary metabolism. The molecular mechanisms linking CL deficiency to these metabolic changes and to the pathologies in BTHS are unknown. We are investigating the role of CL in metabolism using two powerful models. The yeast CL mutant, crd1D, which we generated previously is a well-established model of CL deficiency. More recently, we constructed a Taz knockout mutant, TAZ-KO, in the mouse myoblast C2C12 cell line. TAZ-KO cells exhibit the characteristic biochemical and mitochondrial phenotypes of BTHS and contribute a new model of the disorder. Implementing both models, we have determined that CL regulates two pathways that converge on the TCA cycle - acetyl-CoA synthesis and Fe-S biogenesis. Based on these findings, will use genetic, biochemical, and metabolomic approaches to test the central hypothesis that CL is required for optimal activity of the TCA cycle as a result of its dual role in regulating synthesis of acetyl-CoA and biogenesis of Fe-S cofactors. Aim 1 will define the mechanism whereby CL regulates acetyl-CoA synthesis by increasing the activity of pyruvate dehydrogenase. Aim 2 will characterize anaplerotic mechanisms that rescue TCA cycle deficiencies. Aim 3 proposes to define the role of CL in maturation of yfh1/frataxin, an essential component of the Fe-S machinery. The TCA cycle is a fundamentally important metabolic pathway of carbon metabolism. The current study is driven by a novel hypothesis that identifies a role for CL in regulating the TCA cycle. Elucidating the mechanisms underlying this regulatory role will establish a new paradigm for TCA cycle control. The results may suggest potential new treatments for BTHS and other mitochondrial cardiomyopathies.